Optimization Of Fermentation Conditions Of Kertain-Degarding Bacteria And Research The Related Gene

Keratin, as the main composition of animal feathers, hoof, horn and fur, is widespread in nature. It is difficult to be degraded by general protease because it has a very stable structure, its protein content is very high, if directly discarded it can cause huge waste of protein resources. Keratinase secreted by microorganism can degrade the keratin, which is very important to alleviate the shortage of protein resources in our country, and also is beneficial to environmental protection.The fermentation conditions of keratin-degrading bacteria GZD-23were optimized and keratinase gene produced by strain GZD-23was expressed and the physical and chemical properties of recombinant enzyme were studied.Response surface method was used to optimize fermentation conditions of keratin-degrading bacteria GZD-23. Through the Plackett-Burman experiment, three factors which have significant influence to the production of keratinase in GZD-23were founded, they are sucrose, peptone and pH value. And then by steepest climbing and Box-Behnken experiment, the optimum fermentation conditions for keratinase production of the keratin degrading bacteria GZD-23 were got, they are:sucrose1.6%, peptone0.79%, pH9.17, feather content20g/L, rotate speed100rpm/min, temperature60℃, inoculum size7%, loaded liquid150mL. Fermentating according to the optimum conditions, determined the value of the enzyme activity of keratinase is4.91U/mL, enzyme activity increased104.58%compared to the strain unoptimized fermentation conditions, it was consistented with predicted value of4.96U/mL.The keratinase gene of keratin-degrading bacteria GZD-23was cloned, expressed and purificated. The keratinase gene was amplified through PCR, the length of it is1146bp, encoding382amino acid. The front21amino acid is signal peptide by analysising the amino acid sequence. Then the recombinant expression plasmid PET-30-Ker was constructed and induced.The recombinant keratinase had an approximate molecular mass of44KDa(keratinase39KDa, the label of PET-30a about5KDa), as the same as predict value. At the same time, its expression conditions was optimized, and the optimum condition is:the best induction time8h, the best concentration of IPTG induction0.5mM. Its expression form was mainly inclusion body. Finally, the inclusion body protein was purified, SDS-PAGE showed a single band. Then the pure active keratinase was obtained through gradient dialysis.In the end, the physical and chemical properties of keratinase were studied. The results showed that the optimum temperature of keratinase is60℃, and keratinase was stable within the scope of30～60℃. The optimum pH was8, keratinase was basically stable within the scope of7-9, enzyme activity wasn’t have change much, so keratinase can be stored in this condition. Keratinase activity was promoted under low concentration of Ca2+、Mg2+、 and β-mercaptoethaonl, was inhibited by Cu2+、Mn2+、Zn2+、Co2+、Fe2+、Hg2+、 SDS、TritonX-100and high concentration of β-mercaptoethaonl.Serine protease inhibitors PMSF had a little impact on keratinase activity, however EDTA was strongly inhibited keratinase activity, this suggested that the keratinase may be a kind of matrix metalloproteinases. Furthermore, organic solvents DMSO、 Isopropanol and Acetonitrile inhibited Keratinase activity, the higher the concentration, the greater the inhibition. The research of kinetic characters showed that the values of Km and Vmax were1.15721mg/mL and0.56396ml·min·OD420-1.